Method for producing D-pantothenic acid and/or salts thereof via purification by anion exchange as an additive for animal feed

ABSTRACT

The invention relates to an improved method for producing D-pantothenic acid and/or the salts thereof and to the use thereof as an additive for animal feed.

CROSS REFERENCE TO RELATED APPLICATIONS

The instant application is a U.S. National Stage Application ofInternational Application Number PCT/EP02/01755, filed Feb. 20, 2002.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

Subject matter described and claimed in the instant patent was developedpursuant to a joint research agreement between Omnigene Bioproducts,Inc. and BASF Aktiengesellschaft.

TECHNICAL FIELD

The present invention relates to an improved process for preparingD-pantothenic acid and/or salts thereof and to the use thereof asadditive to animal feedstuffs.

BACKGROUND ART

As a starting material of the biosynthesis of coenzyme A, D-pantothenateis widely distributed in the plant and animal kingdoms. In contrast tohumans who consume sufficient quantities of pantothenic acid via thediet, symptoms of D-pantothenate deficiency are frequently described notonly for plants but also for animals. The availability of D-pantothenateis therefore of great economic interest, particularly in the animal feedindustry.

Conventionally, D-pantothenate is prepared by chemical synthesis fromD-pantolactone and calcium β-alaninate (Ullmann's Encyclopedia ofIndustrial Chemistry, 6^(th) edition, 1999, electronic release, chapter“Vitamins”). The preparation of D-pantolactone requires complex,classical racemate cleavage via dieastereomeric salts. The commercialproduct resulting from the chemical synthesis is usually the calciumsalt of D-pantothenic acid, calcium D-pantothenate.

Compared with chemical synthesis, the advantage of biotechnologicalproduction processes using microorganisms is the selective(enantiomerically pure) production of the D form of pantothenic acid,which can be used for higher organisms. A complex racemate cleavage, asrequired in chemical synthesis, is thus not necessary.

Numerous fermentation processes for preparing D-pantothenic acid usingmicroorganisms are known, including in EP-0 590 857, WO 96/33283, U.S.Pat. No. 6,013,492, WO 97/10340, DE 198 46 499, EP 1 001 027, EP 1 006189, EP 1 006 192 and EP 1 006 193.

Thus EP 1 006 189 and EP 1 001 027 describe processes for preparingpantothenate in which a content of at most 1 g/l of D-pantothenic acidin the fermentation solution is achieved. Such low pantothenic acidcontents in the fermentation solution, that is to say of less than 10%by weight, based on the solids content, are unsuitable, however, foreconomic preparation of D-pantothenic acid-containing animal feedsupplements. A further disadvantage with the processes described to dateis that isolating the product from the fermentation medium requiresnumerous complex work-up steps. An economic preparation process on theindustrial scale is not known.

German Laid Open Application DE 100 16 321 describes a fermentationprocess for preparing a D-pantothenic acid-containing animal feedsupplement. However, an important disadvantage of this process, as withthe above-described fermentation processes for preparing D-pantothenicacid, is that the pantothenic acid precursor β-alanine must be suppliedto the micro-organism via the fermentation medium in order to obtaineconomic yields of the desired product.

In addition, U.S. Pat. No. 6,013,492 and WO 96/332839 describe workingup the D-pantothenic acid from the fermentation solution by filteringoff insoluble constituents (for example cell material) from the culturemedium, adsorbing the filtrate to activated carbon, subsequently elutingthe D-pantothenic acid with an organic solvent, preferably methanol,neutralizing with calcium hydroxide and subsequently crystallizingcalcium D-pantothenate. Important disadvantages are the losses ofvaluable product occurring during crystallization and the use of anorganic solvent which can only be removed with difficulty from theproduct and requires a complex solvent recovery step.

EP 0 590 857 describes a fermentation process for preparingD-pantothenic acid in which culturing a microorganism requires thefeeding of β-alanine. The fermentation solution is filtered to separateoff the biomass, then passed through a cation exchanger and then ananion exchanger, following this neutralizing with calcium hydroxide,concentrating by evaporation, adding activated carbon, filtering oncemore and crystallizing with addition of methanol and calcium chloride.The resultant calcium pantothenate-containing product, in addition toD-pantothenic acid in the form of the calcium salt, also containscalcium chloride in a molar ratio of 1:1. Decreasing the calciumchloride content requires electrodialysis with subsequent spray drying.This process has the disadvantage of being neither economical orecological because of the multiplicity of complex process steps and theuse of organic solvents.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an animal feedsupplement containing D-pantothenic acid and/or salts thereof and itspreparation by an improved process for preparing D-pantothenic acidand/or salts thereof which does not have the abovementioneddisadvantages. For economic reasons, a process is desirable here inwhich supplying β-alanine is greatly decreased or is not required atall. In addition, preparing D-pantothenic acid in the form of itsdivalent salts and, especially, the alkaline earth metal salts, isdesirable, since the divalent salts have less hygroscopiccharacteristics than monovalent salts of pantothenic acid and thus havea less pronounced trend to aggregation for further application, forexample as animal feed supplement.

We have found that this object is achieved advantageously by the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for preparing D-pantothenicacid and/or salts thereof which comprises

-   a) using at least one organism which produces D-pantothenic acid and    in which the biosynthesis of pantothenic acid (pan) and/or    isoleucine/valine (ilv) is deregulated and which forms at least 2    g/l of salts of D-pantothenic acid by fermentation in a culture    medium, 0-20 g/l of free β-alanine and/or β-alanine salt being    supplied to the culture medium,-   b) passing the D-pantothenate-containing fermentation solution    through an anion exchanger,-   c) eluting the D-pantothenate bound to the anion exchanger in the    form of calcium and/or magnesium D-pantothenate using a solution    containing inorganic or organic calcium and/or magnesium salts, or    eluting the bound D-pantothenate in the form of free D-pantothenic    acid using an HCl solution,-   d) optionally adding calcium base and/or magnesium base to set the    free D-pantothenic acid-containing solution to a pH of 3-10, a    solution being obtained which contains calcium and/or magnesium    pantothenate and-   e) subjecting the eluate or the solution containing calcium    pantothenate and/or magnesium pantothenate to drying and/or    formulation.

In a variant of the inventive process, the present process isdistinguished by the fact that in step d), or step e), a suspension isobtained or is charged which contains calcium pantothenate and/ormagnesium pantothenate.

The fermentation taking place in step a) can be carried out usingprocedures which are known per se in the batch, fed-batch or repeatedfed-batch mode or continuously. The resultant pantothenic acid isneutralized in this case using conventional buffer systems, for examplephosphate buffer containing NaOH, KOH or ammonia.

In other variants of the inventive process, in step a) at least 10 g/l,preferably at least 20 g/l, particularly preferably at least 40 g/l,very particularly preferably at least 60 g/l, and in particular at least70 g/l, of salts of D-pantothenic acid are formed in the culture mediumby fermentation.

For the purposes of the present invention, the form of words “producing”means that the organism can synthesize larger amounts of D-pantothenicacid and/or salts thereof than are required for its own metabolic needs.In an inventively advantageous variant, the amount of D-pantothenic acidand/or salts thereof synthesized is not present in the interior of thecell, but ideally is completely released into the culture medium by theorganism. This discharge can be active or passive by means of mechanismswhich are known per se.

According to the invention the D-pantothenic acid-producing organismsused are microorganisms. These include according to the invention fungi,yeasts and/or bacteria. According to the invention, preference is givento using fungi, for example Mucor, or yeasts, for example Saccharomycesor Debaromyces, and of these, preferably, Saccharomyces cerevisiae.Advantageously, coryneform bacteria or Bacillaceae are used according tothe invention. Those which are within the scope of the invention arepreferably, for example, bacteria of the genera Corynebacterium,Escherichia, Bacillus, Arthrobacter, Bevibacterium, Pseudomonas,Salmonella, Klebsiella, Proteus, Acinetobacter or Rhizobium. Particularpreference is given here, for example, to Corynebacterium glutamicum,Brevibacterium breve or Bacillus subtilis, B. licheniformis, B.amyloliquefaciens, B. cereus, B. lentimorbus, B. lentus, B. firmus, B.pantothenticus, B. circulans, B. coagulans, B. megaterium, B. pumilus,B. thuringiensis, B. brevis, B. stearothermophilus and other Bacillusspecies of group 1 which are characterized by their 16sRNA, or Actinummycetalis. This listing serves for explanation and is in no way limitingfor the present invention.

Furthermore, the present invention also comprises the use of geneticallymodified organisms for the inventive preparation of an animal feedsupplement containing free D-pantothenic acid and/or salts thereof. Suchgenetically modified organisms can be isolated, for example, by chemicalmutagenesis and subsequent selection using a suitable “screeningprocess”. According to the invention, what are termed “productionstrains” are also included which are suitable for preparing the productin the meaning of the present invention and have genetic modificationswith respect to the metabolic flux in the direction of D-pantothenicacid, modifications with respect to the discharge of D-pantothenic acidand/or salts thereof via the cell membrane also being included. This canbe achieved, for example, by modifications at key positions in relevantmetabolic biosynthesis pathways of the organism used.

It is also conceivable to use transgenic organisms which result from thetransfer of homologous and/or heterologous nucleotide sequences whichare necessary, or can be required, for synthesizing the desired product.In this case, overexpression and/or deregulation of one or more genesindividually and/or in combination localized in the genome and/or on avector are conceivable.

Transgenic organisms of this type can, advantageously, containadditional copies and/or genetically modified genes selected from thegroup consisting of panB, panC, panD, panE and/or combinations thereofand/or even organization units how contain the panBCD operon. Inaddition, other metabolic pathways, for example the isoleucine-valinebiosynthesis pathway can be advantageously manipulated in the organisms,as is described, for example, in EP 1 006 189, EP 1 006 192, EP 1 006193 or EP 1 001 027. As a result, branched-chain precursor substances ofpantothenic acid biosynthesis are increasingly being made available.Advantageously, if appropriate, the genes for this biosynthesis pathway,i.e. ilvB, ilvN, ilvC and/or ilvD are overexpressed.

In addition, genetic modifications of aspartate α-decarboxylase (panD),for example through overexpression and/or deregulation, in theD-pantothenic acid-producing organism used are covered by the invention.

The word “deregulation”, for the purposes of the present invention,means changing or modifying at least one gene which codes for one enzymein a biosynthetic metabolic pathway, so that the activity of the enzymeis changed or modified in the microorganism. It is preferred that atleast one gene which codes for one enzyme of a biosynthetic metabolicpathway is changed in such a manner that the gene product is formed toan increased extent, or has an increased activity. The term “deregulatedmetabolic pathway” also includes a biosynthetic metabolic pathway inwhich more than one gene, which codes more than one enzyme, is changedor modified in such a manner that the activities of more than one enzymeare changed or modified.

Changes or modifications can include, but are not restricted to:removing the endogenous promoter or regulatory elements; introducingstrong promoters, inducible promoters or a plurality of promoterssimultaneously; removing regulatory sequences, so that expression of thegene product is changed; changing the chromosomal position of the gene;changing the DNA sequence in the vicinity of the gene or within thegene, for example the ribosomal binding site (RBS); increasing thenumber of copies of the gene in the genome or by introducing a varyingnumber of copies of plasmids; modifying proteins (e.g. regulatoryproteins, suppressors, enhancers, transcriptional activators and thelike), which play a role in the transcription of the gene and/or in thetranslation to give the gene product. This also includes all otherpossibilities for deregulating the expression of genes which belong tothe prior art, for example the use of antisense oligonucleotides, or theblocking of repressor proteins.

Deregulation can also comprise changes to the coding region of geneswhich lead, for example, to removing feedback regulation in the geneproduct or to a greater or lesser specific activity of the gene product.

Furthermore, genetic modifications to enzymes are advantageous accordingto the invention which affect the efflux of precursors of pantothenicacid and/or the flux of pantothenic acid to give coenzyme A. Examples ofgenes coding for such enzymes are: alsD, avtA, ilvE, ansB, coaA, coaX,etc. This listing serves for explanation and is in no way limiting forthe present invention.

In addition, genetic modifications are advantageous which secure thecellular production of cofactors (e.g of methylene tetrahydrofolate,redox equivalents and the like) in an amount which is optimum forpantothenic acid production.

Advantageously, thus, β-alanine is already present in the cells inincreased concentrations compared with correspondingly non-geneticallymodified organisms, and thus need not be added to the culture medium asprecursor, as is required, for example, in EP-A 0 590 857.Microorganisms are advantageous in which the biosynthesis of pantothenicacid (pan) and/or isoleucine-valine (ilv) and/orasparate-α-decarboxylase (panD) is deregulated. Furthermore, additionaloverexpression of ketopanthoate reductase (panE) in the microorganismsis advantageous.

It is additionally advantageous according to the invention if, ifappropriate, the coaA gene which is required for the synthesis ofcoenzyme A is decreased in its activity, or is entirely switched off(for example in Bacillus species). This is because Bacillus, in additionto coaA, contains a further gene for this enzymatic function (=coaX).The activity of this gene coaX or the corresponding enzyme can also bechanged, preferably reduced, or even deleted, provided that coaA itselfstill has sufficient enzyme activity, albeit reduced enzyme activity,that is to say the enzyme activity of coaA is not entirely lost. Inaddition to the overexpression of the various genes, geneticmanipulation of the promoter regions of these genes is also advantageousprovided that this manipulation leads to overexpression of the geneproducts.

In an embodiment of the present invention, the bacterial strainsdescribed according to the annex (PCT/US application 0025993), forexample Bacillus subtilis PA 824 and/or derivatives thereof, are used.In a further embodiment, according to the invention the microorganismBacillus subtilis PA 668, as described in the annex (U.S. Ser. No.60/262,995), is used in the inventive process. These strains Bacillussubtilis PA 824 and PA 668 were produced as follows:

Starting from the strain Bacillus subtilis 168 (Marburg strain ATCC6051), which has the genotype trpC2 (Trp⁻), the strain PY79 was producedvia transduction of the Trp⁺ marker (from the Bacillus subtilis wildtype W23). Classical genetic engineering methods (as described, forexample, in Harwood, C. R. and Cutting, S. M. (editors), MolecularBiological Methods for Bacillus (1990) John Wiley & Sons, Ltd.,Chichester, England) introduced mutations ΔpanB and ΔpanE1 into thestrain PY79.

The resultant strain was transformed using genomic DNA of Bacillussubtilis strain PA221 (genotype P₂₆panBCD, trpC2 (Trp⁻)) and genomic DNAof Bacillus subtilis strain PA303 (genotype P₂₆panE1). The resultantstrain PA327 has the genotype P₂₆panBCD, P₂₆panE1, and is a tryptophanauxotroph (Trp⁻). Pantothenic acid titers of up to 3.0 g/l (24 h) wereachieved using Bacillus subtilis strain PA327 in 10 ml culturescontaining SVY medium (25 g/l Difco Veal Infusion Broth, 5 g/l DifcoYeast Extract, 5 g/l of Na glutamate, 2.7 g/l of ammonium sulfatecharged into 740 ml of water, the mixture was autoclaved then 200 ml of1 M potassium phosphate, pH 7.0 and 60 ml of 50% sterile glucosesolution were added), which had been supplemented with 5 g/l ofβ-alanine and 5 g/l of α-ketoisovalerate.

The production of Bacillus subtilis strain PA221 (genotype P₂₆panBCD,trpC2 (Trp⁻)) is described in the following section:

Classic genetic engineering methods were used to clone the panBCD Operonof Bacillus, with the aid of the sequence information of the panBCDOperon of E. coli (see Merkel et al., FEMS Microbiol. Lett., 143,1996:247-252) starting from a Bacillus subtilis GP275 plasmid library.For the cloning, use was made of E. coli strain BM4062 (bir^(ts)) andthe information that the Bacillus operon is close to the birA gene. ThepanBCD operon was introduced into a plasmid which can be replicated inE. coli. To improve the expression of the panBCD operon, strongconstitutive promoters of Bacillus subtilis phages SP01 (P₂₆) were used,and the ribosome binding site (=RBS) before the panB gene was replacedby an artificial RBS. A DNA fragment which is immediately upstream ofthe native panB gene in Bacillus was ligated in front of the P₂₆panBCDcassette on the plasmid. This plasmid was transformed into Bacillussubtilis strain RL-1 (derivative of Bacillus subtilis 168 obtained byclassical mutagenesis (Marburg strain ATCC 6051), genotype trpC2 (Trp⁻))and, by homologous recombination, the native panBCD operon was replacedby the p₂₆panBCD operon. The resultant strain is called PA221 and hasthe genotype P₂₆panBCD, trpC2 (Trp⁻).

A pantothenic acid titer of up to 0.92 g/l (24 h) was achieved using theBacillus subtilis strain PA221 in 10 ml cultures containing SVY mediumwhich had been supplemented with 5 g/l of β-alanine and 5 g/l ofα-ketoisovalerate.

Production of the Bacillus subtilis strain PA303 (genotype P₂₆panE1) isdescribed in the following section:

Using the E. coli panE gene sequence, the Bacillus panE sequence wascloned by analogy. It was found that in B. subtilis, two homologs of theE. coli panE gene exist which were termed panE1 and panE2. Deletionanalyses found that the panE1 gene is responsible for 90% of thepantothenic acid production, while deleting the panE2 gene had nosignificant effect on pantothenic acid production. Here also, similarlyto cloning the panBCD Operon, the promoter was replaced by the strongconstitutive promoter P₂₆ and the ribosome binding site in front of thepanE1 gene was replaced by the artificial binding site. The P₂₆panE1fragment was cloned into a vector which was constructed so that theP₂₆panE1 fragment could integrate into the original panE1 locus in theBacillus subtilis genome. The strain resulting after transformation andhomologous recombination is termed PA303 and has the genotype P₂₆panE1.

A pantothenic acid titer of up to 1.66 g/l (24 h) was achieved using theBacillus subtilis strain PA303 in 10 ml cultures containing SVY mediumwhich had been supplemented with 5 g/l of β-alanine and 5 g/l ofα-ketoisovalerate.

The strain was further constructed by transforming PA327 with a plasmidwhich contained the P₂₆ilvBNC Operon and the marker gene forspectinomycin. The P₂₆ilvBNC operon integrated into the amyE locus,which was demonstrated by PCR. One transformant was termed PA340(genotype P₂₆panBCD, P₂₆panE1, P₂₆ilvBNC, specR, trpC2 (Trp⁻)).

A pantothenic acid titer of up to 3.6 g/l (24 h) was achieved using theBacillus subtilis strain PA340 in 10 ml cultures containing SVY mediumwhich had been supplemented only with 5 g/l of β-alanine; in 10 mlcultures containing SVY medium which had been supplemented with 5 g/l ofβ-alanine and 5 g/l of α-ketoisovalerate, a pantothenic acid titer of upto 4.1 g/l (24 h) was achieved.

In addition, a deregulated ilvD cassette was introduced into strainPA340. For this, a plasmid which contains the ilvD gene under thecontrol of the P₂₆ promoter containing the artificial RBS2 wastransformed into PA340. The P₂₆ilvD gene was integrated into theoriginal ilvD locus by homologous recombination. The resultant strainPA374 has the genotype P₂₆panBCD, P₂₆panE1, P₂₆ilvBNC, P₂₆ilvD, specRand trpC2 (Trp⁻).

A pantothenic acid titer of up to 2.99 g/l (24 h) was achieved using theBacillus subtilis strain PA374 in 10 ml cultures containing SVY mediumwhich had been supplemented only with 5 g/l of β-alanine.

In order to produce pantothenic acid using strain PA374 without feed ofβ-alanine, additional copies of the gene panD coding foraspartate-α-decarboxylase were introduced into strain PA374. For this,chromosomal DNA of strain PA401 was transformed into PA374. Strain PA377was obtained by selection on tetracycline.

The resultant strain PA377 has the genotype P₂₆panBCD, P₂₆panE1,P₂₆ilvBNC, P₂₆ilvD, specR, tetR and trpC2 (Trp⁻).

A pantothenic acid titer of up to 1.31 g/l (24 h) was achieved withoutfeed of precursors using Bacillus subtilis strain PA377 in 10 mlcultures containing SVY medium.

Preparation of Bacillus subtilis strain PA401 (genotype P₂₆panD) isdescribed in the following section:

The Bacillus subtilis panD gene was cloned from the panBCD operon into avector which carries the tetracycline marker gene. The promoter P₂₆ andan above-described artificial RBS were cloned in front of the panD gene.Restriction digestion produced a fragment which contained thetetracycline marker gene and the P₂₆panD gene. This fragment wasreligated and transformed into the above-described strain PA221. Thefragment integrated into the genome of strain PA211. The resultantstrain PA401 has the genotype P₂₆panBCD, P₂₆panD, tetR and trpC2 (Trp⁻).

A pantothenic acid titer of up to 0.3 g/l (24 h) was achieved using theBacillus subtilis strain PA401 in 10 ml cultures containing SVY mediumwhich had been supplemented with 5 g/l of α-ketoisovalerate. In 10 mlcultures containing SVY medium which had been supplemented with 5 g/l ofD-pantoic acid and 10 g/l of L-aspartate, pantothenic acid titers of upto 2.2 g/l (24 h) were achieved.

Starting from strain PA377, a tryptophan-prototrophic strain wasgenerated by transformation with chromosomal DNA from strain PY79. Thisstrain PA824 has the genotype P₂₆panBCD, P₂₆panE1, P₂₆ilvBNC, P₂₆ilvD,specR, tetR and Trp⁺.

A pantothenic acid titer of up to 4.9 g/l (48 h) without supply ofprecursors was achieved using Bacillus subtilis strain PA824 in 10 mlcultures in SVY medium (comparison PA377: up to 3.6 g/l in 48 h).

The preparation of PA668 is described in the following section:

The Bacillus panB gene was cloned from the wild type panBCD operon andinserted into a vector which, in addition to a chloramphenicolresistance gene, also contains B. subtilis sequences of the vpr locus.

The strong constitutive promoter P₂₆ was introduced before the 5′ end ofthe panB gene. One fragment which contains the P₂₆panB gene, the markergene for chloramphenicol resistance and also Bacillus subtilis vprsequences was obtained by restriction digestion. The isolated fragmentwas religated and used to transform strain PA824. The resultant strainwas termed PA668. The genotype of PA668 is: P₂₆panBCD, P₂₆panE1,P₂₆ilvBNC, P₂₆ilvD, P₂₆panB, specR, tetR, CmR and Trp⁺.

Two colonies of PA668 were isolated and termed PA668-2A, and the otherPA668-24.

Using B. subtilis strain PA668-2_ pantothenic acid titers of 1.5 g/l areachieved in 48 h in 10 ml cultures in SVY medium without supply ofprecursors. In 10 ml cultures supplemented with 10 g/l of aspartate,titers up to 5 g/l are achieved.

Using B. subtilis strain PA668-24, pantothenic acid titers of 1.8 g/lare achieved in 48 h in 10 ml cultures in SVY medium without supply ofprecursors. In 10 ml cultures supplemented with 10 g/l of L-aspartate,titers up to 4.9 g/l are achieved.

The exact construction of the strains is to be taken from the annexes(PCT/US application 0025993 and U.S. Ser. No. 60/262,995).

Using the above-described strain PA377, in glucose-limited fermentationin SVY medium (25 g/l of Difco Veal Infusion Broth, 5 g/l of Difco YeastExtract, 5 g/l of tryptophan, 5 g/l of Na glutamate, 2 g/l of (NH₄)₂SO₄,10 g/l of KH₂PO₄, 20 g/l of K₂HPO₄, 0.1 g/l of CaCl₂, 1 g/l MgSO₄, 1 g/lof sodium citrate, 0.01 g/l of FeSO₄.7H₂O and 1 ml/l of a trace saltsolution of the following composition: 0.15 g of Na₂MoO₄.2H₂O, 2.5 g ofH₃BO₃, 0.7 g of CoCl₂.6H₂O, 0.25 g of CuSO₄.5H₂O, 1.6 g of MnCl₂.4H₂O,0.3 g of ZnSO₄.7H₂O, made up to 1 l with water)) on a 10 l scale withcontinuous supply of a glucose solution, pantothenic acid concentrationsin the fermentation broth of 18-19 g/l 122-25 g/l) are achieved in 36 h(48 h).

In the case of glucose-limited fermentation of PA824, thetryptophan-prototroph derivative of PA377, in yeast extract medium (10g/l of Difco Yeast Extract, 5 g/l of Na glutamate, 8 g/l of (NH₄)₂SO₄,10 g/l of KH₂PO₄, 20 g/l of K₂HPO₄, 0.1 g/l of CaCl₂, 1 g/l of MgSO₄, 1g/l of sodium citrate, 0.01 g/l of FeSO₄.7H₂O and 1 ml/l of theabove-described trace salt solution) the following pantothenic acidconcentrations in fermentation broths are achieved in 36 h, 48 h and 72h: 20 g/l, 28 g/l and 36 g/l, on a 10 l scale with continuous supply ofa glucose solution.

By means of further optimization of media, using strain PA824 inglucose-limited fermentation in a medium consisting of 10 g/l of DifcoYeast Extract, 10 g/l of NZ amine A (Quest International GmbH,Erftstadt), 10 g/l of Na glutamate, 4 g/l of (NH₄)₂SO₄, 10 g/l ofKH₂PO₄, 20 g/l of K₂HPO₄, 0.1 g/l of CaCl₂, 1 g/l of MgSO₄, 1 g/l ofsodium citrate, 0.01 g/l of FeSO₄. 7H₂O and 1 ml/l of theabove-described trace salt solution, pantothenic acid concentrations of37 g/l (48 g/l) are achieved in fermentation broths in 36 h (48 h) on a10 l scale with continuous supply of a glucose solution.

Further increases in the pantothenic acid concentration in thefermentation broth are conceivable by further optimization of media, byincreasing the fermentation time, by process and strain improvement andby combinations of the individual steps. Thus the above-describedpantothenic acid concentrations are also achievable by fermentation ofstrains which are derivatives of the above-described PA824. Derivativescan be prepared by classical strain development and by further geneticengineering manipulations. By development of media, strain andfermentation process, the pantothenic acid titers in the fermentationbroths can be increased to greater than 40, 45, 50, 55, 60, 65, 70, 75,80, 85 and >90 g/l.

An essential advantage of the inventive process is that the fermentationis carried out in a culture medium which, apart from at least one carbonsource and nitrogen source, contains no other precursors as startingcompounds. That is to say the biosynthesis of D-pantothenic acid isindependent of the supply of other precursors. For the purposes of thepresent invention, such precursors are substances such as β-alanineand/or L-aspartate and/or L-valine and/or α-ketoisovalerate and/orcombinations thereof.

In a preferred variant of the inventive process, the fermentation of theD-pantothenic-acid-producing organism is carried out in a culture mediumwhich contains a carbon source and a nitrogen source, but to which nofree β-alanine and/or β-alanine salts is/are added or supplied in thecourse of the fermentation. That is to say for producing D-pantothenicacid in ranges of at least 10 g/l of culture medium, preferably at least20 g/l, particularly preferably at least 40 g/l, very particularlypreferably at least 60 g/l, and in particular at least 70 g/l, no supplyof free β-alanine and/or β-alanine salts is required according to theinvention.

Independence from feed of precursors is in particular an importanteconomic advantage of the inventive process compared with knownprocesses, since a multiplicity of precursors are very expensive.

However, the invention does not exclude addition of β-alanine and/orβ-alanine salts, so that therefore the yield of D-pantothenic acid canbe further increased by adding β-alanine and/or β-alanine salts. If itis assumed, for example, that all of the required precursors ofpantothenic acid are present in a sufficient amount, only the activityof the panD gene limits a further increase in pantothenic acidproduction, then the yield of pantothenic acid can be increased, forexample, by a further 50% by adding free β-alanine and/or β-alaninesalts.

In an advantageous variant of the present invention, up to 20 g/l offree β-alanine and/or β-alanine salts can be added to the culture mediumfor additional increase in the pantothenic acid yield by more than 50%.Preference is given to adding about 15 g/l of free β-alanine and/orβ-alanine salts to the culture medium.

Examples of carbon sources which are suitable according to the inventionfor use in a culture medium for fermenting the abovementioned organismsare sugars, such as starch hydrolysates (mono-, di-, oligosaccharides),preferably glucose or sucrose, and also beet or cane sugar molasses,proteins, protein hydrolysates, soybean meal, corn steep liquor, fats,free fatty acids, recirculated cells from previous fermentations orhydrolysates thereof, and also yeast extract. This listing is notlimiting for the present invention.

In addition, the present process is advantageously distinguished in thatthe total sugar content is reduced to a minimum up to the end offermentation, since this would otherwise make difficult later dryingand/or formulation of the fermentation solution owing to sticking. Thiscan be achieved according to the invention by continuing thefermentation for some further time after the carbon source is consumed(in the case of batch culture) or after the carbon feed (in the case ofa process procedure in the fed-batch or repeated fed-batch mode) isinterrupted and/or regulated in such a manner that the concentration ofthe carbon source is virtually zero (in the case of fed-batch, repeatedfed-batch or continuous process procedure).

This is achieved according to the invention by the means that, afterinterrupting the addition of the carbon source (for example sugarsolution), the fermentation is continued until the dissolved oxygenconcentration (pO₂) of at least 80%, preferably 90%, and particularlypreferably 95%, of the saturation value is achieved in the fermentationsolution.

Examples of nitrogen sources which are suitable according to theinvention are ammonia, ammonium sulfate, urea, proteins, proteinhydrolysates or yeast extract. This listing also is not limiting for thepresent invention.

In addition, the fermentation medium contains mineral salts and/or traceelements, such as amino acids and vitamins. The exact compositions ofsuitable fermentation media are known in abundance and accessible tothose skilled in the art.

After the fermentation medium has been inoculated with a suitableD-pantothenic-acid-producing organism (at the cell densities known tothose skilled in the art), if appropriate with addition of an antifoam,the organism is cultured. Any necessary regulation of the pH of themedium can be achieved using various inorganic or organic alkalis oracids, for example NaOH, KOH, ammonia, phosphoric acid, sulfuric acid,hydrochloric acid, formic acid, succinic acid, citric acid or the like.

On account of the buffer systems used during fermentation, which, asdescribed above, can be, for example, NaOH, KOH, ammonia, phosphoricacid, sulfuric acid, hydrochloric acid, formic acid, succinic acid,citric acid or the like, the D-pantothenic acid formed is present in thefermentation solution, depending on the buffer system used, in the formof the respective salt(s). Since in this case, in particular, the saltsof D-pantothenic acid in the form of their monovalent cations aredisadvantageous, the fermentation solution is prepared according to theinvention with the use of an anion exchanger.

The present invention comprises here all commercially available anionexchangers. These include all strongly or weakly basic resins;preference is given to the strongly basic resins. It is advantageousaccording to the invention to use the following anion exchangers, thelisting serving only to describe the present invention and not to limitit: Lewatit M500, MP 500 from Bayer, Amberlite IRA 402, IRA 410, IRA 900from Rohm & Haas, Dowex 1×2, 1×8 from Dow Chemicals, Diaion PA 306, PA316, PA 406, PA412 or PA 418 from Mitsubishi Chemicals Corporation, andLewatit MP 62 from Bayer, MWA-1 from Dow Chemicals, Amberlite IRA 67,IRA 96SB, IRA 96 RF, Duolite A561 or A7 from Rohm & Haas, Reillex 402from Reilly Industries, Diaion WA 21 or WA 30 from Mitsubishi ChemicalsCorporation.

Using the anion exchanger bind the D-pantothenate and other anions fromthe fermentation solution, while the remaining compounds, in particularmonovalent cations, for example ammonium, potassium or sodium, passthrough the anion exchanger and are then discarded. In this manner theunwanted cations are advantageously virtually completely removed fromthe fermentation solution.

The D-pantothenate bound to the anion exchanger is then eluted in theform of calcium and/or magnesium D-pantothenate by a solution containinginorganic or organic calcium and/or magnesium salts, or is eluted in theform of free D-pantothenic acid by an HCl solution. According to theinvention the salts can be acidic and/or basic inorganic and/or organiccalcium salts and/or magnesium salts. For instance, in a variant of theinventive process of the present invention, it is conceivable to elutethe D-pantothenate bound to the anion exchanger by a solution containingacidic inorganic and/or organic calcium salts and/or magnesium salts.Likewise, a variant is conceivable in which the elution solution in stepc) contains inorganic and/or organic calcium bases and/or magnesiumbases. The basic calcium salts and/or magnesium salts can be presentinstead of, or in addition to, the acidic calcium salts and/or magnesiumsalts. In further variants of the present invention, the elutionsolution in step c) of the inventive process can contain inorganiccalcium salts and/or magnesium salts, acidic organic calcium saltsand/or magnesium salts, in organic calcium bases and/or magnesium basesand/or organic calcium bases and/or magnesium bases and correspondingcombinations thereof.

Advantageously, for the purposes of the present invention, inorganiccalcium salts and/or magnesium salts are the corresponding halides.Preferably, according to the invention, these are CaCl₂ and/or MgCl₂.Examples of inorganic calcium bases and/or magnesium bases are calciumhydroxide, calcium carbonate, calcium oxide, magnesium hydroxide ormagnesium carbonate. For the purposes of the present invention, theorganic calcium salts and/or magnesium salts are, for example, highlywater-soluble salts of organic anions. Preferably, these are, forexample, calcium and/or magnesium formate, acetate, propionate,glycinate and/or lactate.

In a variant of the present invention, the D-pantothenate bound to theanion exchanger is eluted with a 5-10% strength CaCl₂ solution, MgCl₂solution or aqueous HCl solution, the eluate containing a D-pantothenatein the form of calcium or magnesium D-pantothenate, or as freeD-pantothenic acid.

If the elution is performed with an aqueous HCl solution, the freeD-pantothenic-acid-containing eluate is adjusted to a pH of 3-10 byadding calcium base and/or magnesium base. A pH of 5-10 is advantageous.Preferably, the solution is adjusted to a pH of 5-9, particularlypreferably 6-9, and very particularly preferably 6-8. In this manner asolution is obtained which contains calcium pantothenate and/ormagnesium pantothenate.

After adding calcium base and/or magnesium base to adjust the pH of theeluate containing free D-pantothenic acid, according to the invention asuspension can also be obtained which contains calcium pantothenateand/or magnesium pantothenate.

Preferably, for neutralization, calcium hydroxide, calcium carbonate,calcium oxide, magnesium hydroxide and/or basic magnesium carbonate isadded to the eluate containing free D-pantothenic acid in the form of asolid and/or as aqueous suspension.

It is preferred according to the invention in this case if the eluatecontaining free D-pantothenic acid is neutralized with a calcium baseand/or magnesium base in the form of an aqueous suspension. As a resultof using such an aqueous suspension the neutralization is performed morerapidly and without relatively large pH fluctuations than is the casewhen a corresponding solid is used.

According to the invention the process is distinguished in that anaqueous suspension comprising 2-55% by weight, preferably 10-50% byweight, and particularly preferably 20-40% by weight, of calciumhydroxide is added to the eluate optionally in step d).

The invention additionally comprises a process in which an aqueoussuspension comprising 2-65% by weight, preferably 10-50% by weight, andparticularly preferably 20-40% by weight, of calcium carbonate is addedto the eluate optionally in step d). In a further embodiment of thepresent invention, an aqueous suspension comprising 2-60% by weight,preferably 10-50% by weight, and particularly preferably 20-40% byweight, of magnesium hydroxide is added to the eluate optionally in stepd) of the inventive process. The invention also comprises a process inwhich an aqueous suspension comprising 2-25% by weight, preferably10-20% by weight, of basic magnesium carbonate is added to the eluateoptionally in step d).

In the eluate or the solution or the suspension containing calciumD-pantothenate and/or magnesium D-pantothenate resulting from steps c)or d) of the inventive process, in a manner described above, by using ananion exchanger, the content of monovalent cations, preferably ammonium,potassium and/or sodium ions, is decreased to a concentration of ≦1 g/kgof eluate/solution.

The eluate or the solution or suspension containing calcium and/ormagnesium pantothenate is dried and/or formulated using processes knownper se, for example spray drying, spray granulation, fluidized-beddrying, fluidized-bed granulation, drum drying or spin-flash drying(Ullmann's Encyclopedia of Industrial Chemistry, 6^(th) edition, 1999,electronic release, chapter “Drying of Solid Materials”). The gas inlettemperature in convection drying is in the range 100-280° C., preferably120-210° C. The gas outlet temperature is 50-180° C., preferably 60-150°C. To establish a desired particle size distribution and the associatedproduct properties, fine particles can be separated off andrecirculated. In addition, course material can be ground in a mill andlikewise then recirculated.

According to the invention, in the process described above, thereduction of complex workup steps is advantageous, in particular theavoidance of the use of organic solvents, with simultaneous productionof a desired product having high biological value. In addition,according to the invention the amount of waste water produced issignificantly reduced. This thus results in further savings in complexwork up and disposal plants. Thus the inventive process isadvantageously distinguished in that it is simpler, less susceptible tofaults, less time-consuming, markedly less expensive and thus moreeconomical than conventional processes.

However, this does not exclude the inventive process from being able tobe varied. The inventive process steps a) to d) mentioned at the outsetcan be supplemented by one or more of the following process steps whichare themselves familiar to those skilled in the art. In this case, allconceivable combinations of the additional process steps with theprocess steps a) to e) are covered by the invention.

Thus the solutions or suspensions resulting from the process steps a)-d)can be disinfected, for example by heating (sterilization) or othermethods, for example pasteurization or sterile filtration.

In other variants of the inventive process, before the drying and/orformulation of the solution or suspension, at least one of and/orcombinations of the following steps can be carried out, comprising lysisand/or sterilizing the biomass and/or separating off the biomass fromthe fermentation solution and/or adding further additives and/orconcentrating the fermentation solution, preferably by removing water.

The present invention thus also relates to a process in which the lysisand/or sterilization of the biomass is carried out still in thefermentation solution or not until after the biomass is separated offfrom the fermentation solution. This can be performed, for example, by atemperature treatment, preferably at 80-200° C., and/or an acidtreatment, preferably with sulfuric acid or hydrochloric acid, and/orenzymatically, preferably with lysozyme.

In a further embodiment of the present invention, the cells of thefermented microorganisms can be removed by filtration, separation (forexample centrifugation) and/or decantation from the solutions orsuspensions of the steps a), c) or d) of the inventive process. It isalso conceivable that the solution from step a) are passed directlythrough an anion exchanger without separating off the organisms present.

If the biomass is not separated off before the workup step via anionexchangers (step b)) of the inventive process, the biomass-containingfermentation solution can also advantageously be passed through theion-exchange bed from bottom to top, that is to say in the oppositedirection to gravity.

The solution resulting from the work up via the anion exchanger can,optionally following neutralization, be concentrated via a suitableevaporator, for example falling-film evaporator, thin-film evaporator orrotary evaporator. Such evaporators are manufactured, for example, bythe companies GIG (4800 Attnang Puchheim, Austria), GEA Canzier (52303Düren, Germany), Diessel (31103 Hildesheim, Germany) and Pitton (35274Kirchhain, Germany).

To improve the color properties of the end product, an additionalfiltration step can be carried out in which a little activated carbon isadded to the solutions or suspensions obtained during the process andthe resultant suspension is then filtered. Or, the solutions obtainedduring the fermentation can be passed through a small activated carbonbed. The amounts of activated carbon used which are required for thisare in the range of a few % by weight of the solution and are within theknowledge and experience of those skilled in the art.

These filtrations can be simplified by adding a commercial flocculatingaid to the respective solution or suspension before filtration (forexample Sedipur CF 902 or Sedipur CL 930 from BASF AG, Ludwigshafen).

In an advantageous embodiment of the present invention, the fermentationoutput (fermentation broth) is sterilized by heating and is then freedfrom the cell mass by centrifugation, filtration or decantation. Afteraddition of 50-1000 mg/kg, preferably 100-200 mg/kg, of a commerciallyconventional flocculating aid, based on the fermentation output, themixture is filtered through a short bed of activated carbon and sand inorder to obtain a biomass-free solution having a high D-pantothenic acidcontent. This treated solution is then passed through the ion-exchangebed.

If the biomass is not separated off before the inventive workup step viaan anion exchanger, the biomass-containing fermentation solutionadvantageously can also be passed through the ion-exchange bed frombottom to top, that is to say in the opposite direction to gravity. Thisprocedure is generally advantageous if suspended matter is present inthe solution or suspension to be purified.

This solution or suspension can then be dried, for example by spraydrying. This can be performed in cocurrent, countercurrent or mixedflow. For the atomization, all known atomizers can be used, inparticular centrifugal atomizers (atomizer disk), single-fluid nozzle ortwo-fluid nozzle. Preferred drying temperature conditions are 150-250°C. tower inlet temperature and 70-130° C. tower exit temperature.However, drying can also be performed at higher or lower temperaturelevels. To achieve a very low residual moisture, a further drying stepcan be provided downstream in a fluidized bed.

The spray drying may also be carried out in an FSD or SBD dryer (FSD:fluidized spray dryer; SBD: spray bed dryer), as are manufactured by thecompanies Niro (Copenhagen, Denmark) and APV-Anhydro (Copenhagen,Denmark), which are a combination of spray dryer and fluidized bed.

In the spray drying an anticaking agent can be added. This can reducethe deposition on the dryer wall and improve the flow behavior,precisely in the case of fine-grained powders. Anticaking agents whichcan be used are, in particular, silicates, stearates, phosphates andcorn starch.

In principle the drying can also take place in a sprayed fluidized bed,in which case this can be operated not only continuously but alsobatchwise. The solution or suspension can be sprayed in not only fromthe top (top spray) and from the bottom (bottom spray) but also from theside (side spray).

The present invention further relates to a composition for use as animalfeed additive and/or animal feed supplement, in which case it can beprepared by

-   a) using at least one organism which produces D-pantothenic acid and    in which the biosynthesis of pantothenic acid (pan) and/or    isoleucine/valine (ilv) is deregulated and which forms at least 2    g/l of salts of D-pantothenic acid by fermentation in a culture    medium, 0-20 g/l, preferably 0 g/l, of free β-alanine and/or    β-alanine salt being supplied to the culture medium,-   b) passing the D-pantothenate-containing fermentation solution    through an anion exchanger,-   c) the D-pantothenate bound to the anion exchanger is eluted in the    form of calcium and/or magnesium D-pantothenate using a solution    containing inorganic or organic calcium salts and/or magnesium    salts, or is eluted in the form of free D-pantothenic acid using an    HCl solution,-   d) optionally adding calcium base and/or magnesium base to set the    free D-pantothenic acid-containing eluate to a pH of 3-10, a    solution being obtained which contains calcium and/or magnesium    pantothenate acid and-   e) subjecting the eluate or the solution containing calcium    pantothenate and/or magnesium pantothenate to a drying and/or    formulation.

In a variant of the present invention, in step c), an elution solutioncan be used which contains acidic inorganic and/or organic calciumand/or magnesium salts and/or basic inorganic and/or organic calciumand/or magnesium salts.

In a further variant of the present invention, in step d) or in step e),a suspension can be obtained or charged which contains calciumpantothenate and/or magnesium pantothenate and results in the inventivecomposition after the further processing.

According to the invention the composition is distinguished in that itcomprises salts of D-pantothenic acid at a concentration of at least1-100% by weight, preferably at least 20-100% by weight, andparticularly preferably at least 50% by weight. The present inventionrelates to a composition which comprises salts of D-pantothenic acid inthe form of divalent cations, preferably calcium and/or magnesiumD-pantothenate. According to the invention preference is given to acomposition which is distinguished in that the content of salts ofD-pantothenic acid in the form of monovalent cations is ≦1 g/kg.

According to the invention by means of the above-described process acalcium D-pantothenate and/or magnesium D-pantothenate is obtained whichmeets the requirements for a feed additive. These requirements are, forexample, a relatively high content of D-pantothenate and a highcompatibility with the target organism and biological value in themeaning of “vitamin activity” of the inventive product.

The present invention will be described in more detail by the followingexamples, which are not, however, limiting for the invention:

MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1

In a laboratory fermenter containing stirrer and gas-introduction deviceof 14 l capacity, aqueous fermentation medium of the followingcomposition is charged:

Concentration Starting material [g/l] Yeast extract 10 Sodium glutamate5 Ammonium sulfate 8 KH₂PO₄ 6.7 K₂HPO₄ 9.8

After sterilization, the following sterile media components areadditionally added:

Concentration Starting material [g/l] Glucose 2.5 Calcium sulfate 0.1Magnesium sulfate 1 Sodium citrate 1 FeSO₄.7H₂O 0.01 Trace salt solution1 ml

The trace salt solution has the following composition:

0.15 g of Na₂MoO₄.2H₂O, 2.5 g of H₃BO₃, 0.7 g of CoCl₂.6H₂O, 0.25 g ofCuSO₄.5H₂O, 1.6 g of MnCl₂.4H₂O, 0.3 g of ZnSO₄.7H₂O are made up to 1 lwith water. The trace salt solution is added via sterile filtration. Theinitial liquid volume is 6 l. The contents set forth above are based onthis value.

To this solution are added 60 ml of inoculation culture (OD=10 ) ofBacillus subtilis PA824 and the suspension is fermented at 43° C. withvigorous stirring at a gas introduction rate of 12 l/min. This strain isdescribed in accordance with the annex in PCT/US Application 0025993.

Within the course of 47 h, 6.5 l of a sterile aqueous solution areadded, the composition of which is as follows:

Concentration Starting material [g/l] Glucose 550 Calcium sulphate 0.7Trace salt solution 6 ml

The fermentation is carried out under glucose-limiting conditions.During the fermentation the pH is regulated to 7.2 by adding 25%strength ammonia solution or 20% strength phosphoric acid. Ammoniaserves simultaneously as nitrogen source for the fermentation. The speedof rotation of the agitator element is controlled to keep the dissolvedoxygen content to 30% of the saturation value. After halting theaddition of the carbon source, the fermentation is continued until thedissolved oxygen content (pO₂) has reached a value of 95% of thesaturation value. The fermentation is then ended and the organism isthermally destroyed. For this the fermentation solution is sterilizedfor 45 min. Successful destruction is demonstrating by plating out.

The cells are then separated off by centrifugation. After cellseparation the concentration of D-pantothenate after 48 h is 22.8 g/l.

Similarly, fermentation broths may also be produced which haveβ-alanine-feed-free pantothenic acid titers of greater than 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and >90 g/l.

1000 ml of the fermentation broth are pumped from bottom to top througha 250 ml glass column in which are situated approximately 100 ml of thestrongly basic ion exchanger Lewatit MP 500 (in the OH⁻ form). The flowrate is approximately 20 ml/min. The column is then washed at the samepumping rate with approximately 1000 ml of deionized water. Thissolution contains cations in the form of NH4⁺, Ca² ⁺, K⁺, Mg²⁺ and Na⁺ions. The D-pantothenic acid bound to the ion exchanger is then elutedusing approximately 1000 ml of a 10% strength calcium chloride solution.The eluate contains pantothenic acid in the form of calciumD-pantothenate, sulfate and phosphate ions of differing valencies. A 75ml sample of the resultant calcium D-pantothenate solution was thendried by evaporating off the water on a rotary evaporator and 4.8 g of alight-brown calcium D-pantothenate powder are obtained which has acontent of 35% calcium D-pantothenate. This powder has no tendency tostick together and has good product properties.

EXAMPLE 2

In a laboratory fermenter containing stirrer and gas-introduction deviceof 14 l capacity, aqueous fermentation medium of the followingcomposition is charged:

Concentration Starting material [g/l] Yeast extract 10 Sodium glutamate5 Ammonium sulfate 8 KH₂PO₄ 8.4 K₂HPO₄ 15

After sterilization, the following sterile media components areadditionally added:

Concentration Starting material [g/l] Glucose 2.5 Calcium chloride 0.1Magnesium chloride 1 Sodium citrate 1 FeSO₄.7H₂O 0.01 Trace saltsolution 1 ml

The trace salt solution has the following composition:

0.15 g of Na₂MoO₄.2H₂O, 2.5 g of H₃BO₃, 0.7 g of CoCl₂.6H₂O, 0.25 g ofCuSO₄.5H₂O, 1.6 g of MnCl₂.4H₂O, 0.3 g of ZnSO₄.7H₂O are made up to 1 lwith water. The trace salt solution is added via sterile filtration. Theinitial liquid volume is 5.5 l. The contents set forth above are basedon this value.

To this solution are added 55 ml of inoculation culture (OD=10) ofBacillus subtilis PA824 and the suspension is fermented at 43° C. withvigorous stirring at a gas introduction rate of 12 l/min. This strain isdescribed in accordance with the annex in PCT/US Application 0025993.

Within the course of 48 h, 6 l of a sterile aqueous solution are added,the composition of which is as follows:

Concentration Starting material [g/l] Glucose 550 Calcium chloride 0.6Trace salt solution 6 ml

The fermentation is carried out under glucose-limiting conditions.During the fermentation the pH was regulated to about 7.2 by adding 25%strength ammonia solution or 20% strength phosphoric acid. Ammoniaserves simultaneously as nitrogen source for the fermentation. The speedof rotation of the agitator element is controlled to keep the dissolvedoxygen content to 30% of the saturation value. After halting theaddition of the carbon source, the fermentation is continued until thedissolved oxygen content (pO₂) has reached a value of 95% of thesaturation value. The fermentation is then ended and the organism isthermally destroyed. For this the fermentation solution is sterilizedfor 30 min. Successful destruction is demonstrating by plating out.

The cells are then separated off by centrifugation. After cellseparation the concentration of D-pantothenate on stopping after 48 h is24.1 g/l.

Similarly, fermentation broths may also be produced which haveβ-alanine-feed-free pantothenic acid titers of greater than 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and >90 g/l.

1300 ml of the fermenter discharge were transported through a bed(volume about 1 liter) of the ion exchanger Lewatit MP 500 (in the Cl⁻form) and washed out with about 1 l of water. The flow rate is regulatedto approximately 20 ml/min.

At the same flow rate, 2 l of a 5% strength milk of lime suspension werepumped through the ion exchanger. A solution was eluted which had acalcium D-pantothenate content of 28 g/l. Cations in the form ofammonium, potassium, magnesium or sodium ions were not detected. Thephosphate ion concentration had been reduced by 20% in comparison withthe starting solution before the ion exchanger.

This aqueous calcium D-pantothenate solution was dried in a Niro Minorspray dryer. The tower inlet temperature was about 200° C., and thetower outlet temperature 85-90° C. The atomization was performed using atwo-fluid nozzle at a pressure of 4 bar. Powdering agent was not added.

The pulverulent product had a specification of (data in % by weight):

-   Water content: 2 g/100 g-   Calcium D-pantothenate: 56.3 g/100 g-   Ammonium: <0.01 g/100 g-   Potassium: <0.01 g/100 g-   Sodium: <0.01 g/100 g-   Magnesium: <0.01 g/100 g

EXAMPLE 3

In a laboratory fermenter containing stirrer and gas-introduction deviceof 300 l capacity, aqueous fermentation medium of the followingcomposition is charged:

Concentration Starting material [g/l] Soybean meal 40 Yeast extract 5Sodium glutamate 5 Ammonium sulfate 8 KH₂PO₄ 10 K₂HPO₄ 20

After sterilization, the following sterile media components areadditionally added:

Concentration Starting material [g/l] Glucose 2.5 Calcium sulfate 0.1Magnesium sulfate 1 Sodium citrate 1 FeSO₄.7H₂O 0.01 Trace salt solution1 ml

The trace salt solution has the following composition:

0.15 g of Na₂MoO₄.2H₂O, 2.5 g of H₃BO₃, 0.7 g of CoCl₂.6H₂O, 0.25 g ofCuSO₄.5H₂O, 1.6 g of MnCl₂.4H₂O, 0.3 g of ZnSO₄.7H₂O are made up to 1 lwith water. The trace salt solution is added via sterile filtration. Theinitial liquid volume is 100 l. The contents set forth above are basedon this value.

To this solution are added 4 l of inoculation culture (OD=120) ofBacillus subtilis PA824 and the suspension is fermented at 43° C. withvigorous stirring at a gas introduction rate of 1.8 m³/h. This strain isdescribed in accordance with the annex in PCT/US Application 0025993.

Within the course of 43 h, 113 l of a sterile aqueous solution areadded, the composition of which is as follows:

Concentration Starting material [g/l] Glucose 550 Calcium chloride 0.6Sodium citrate 2 FeSO₄.7H₂O 0.02 Sodium glutamate 5 Trace salt solution1 ml

The fermentation is carried out under glucose-limiting conditions.During the fermentation the pH is regulated to 7.2 by adding 25%strength ammonia solution or 20% strength phosphoric acid. Ammonia actssimultaneously as nitrogen source for the fermentation. The speed ofrotation of the agitator element is controlled to keep the dissolvedoxygen content to 30% of the degree of saturation. After halting theaddition of the carbon source, the fermentation is continued until thedissolved oxygen content (pO₂) has reached a value of 95% of thesaturation value. After 43 h, the fermentation is ended. The cells areremoved by separating in a disk centrifuge. The cell-free fermentationsolution is then sterilized for 60 min. Successful destruction isdemonstrated by plating out.

The concentration of D-pantothenate after cell separation andsterilization is 21 g/l. Similarly, fermentation broths may also beproduced which have β-alanine-feed-free pantothenic acid titers ofgreater than 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85and >90 g/l. The fermentation solution was filtered.

Of the resultant filtrate, 1800 ml were passed through a bed (1 liter)of the ion exchanger Lewatit MP500 (in the OH⁻ form). The ion exchangerwas washed with water. The flow rate was approximately 20 ml/min.

At the same flow rate, approximately two liters of an 8% strengthhydrochloric acid solution were passed through the ion exchanger. Theeluate was collected and a pH of approximately 7 was set using 20% milkof lime (slurry of calcium hydroxide in water). The eluate was thenfiltered again through a paper filter and the filtrate obtained was 2174g of an aqueous calcium D-pantothenate solution having a calciumD-pantothenate content of 21 g/l.

The spray drying was performed in a Niro Minor laboratory spray dryer.The tower inlet temperature was a mean 185° C., and the tower outlettemperature approximately 95° C. The atomization was performed using atwo-fluid nozzle at a pressure of 2 bar. A pulverulent product of thefollowing specification was obtained (data in % by weight):

-   Water content: 1.4 g/100 g-   Calcium D-pantothenate: 52.2 g/100 g-   Ammonium: <0.01 g/100 g-   Potassium: <0.01 g/100 g-   Sodium: <0.01 g/100 g-   Magnesium: <0.01 g/100 g

EXAMPLE 4

In a fermenter equipped with stirrer and gas-introduction device of 20 1capacity, 200 g of soybean meal, 50 g of 50% strength yeast extract, 25g of Na glutamate, 40 g of ammonium sulfate and 5 ml of antifoam agentTego KS911 were admixed with 3.9 l of deionized water and the contentswere sterilized at 121° C. for 30 min.

Solutions 1, 2, 3 and 4 were then added.

Solution 1 was made up as follows: 87.5 g of glucose, 0.5 g ofCaCl₂.2H₂O and 5 g of MgCl₂.6H₂O were dissolved in 500 ml of deionizedwater and sterilized.

Solution 2 was made up as follows: 25 ml of citrate-iron solution (200g/l of sodium citrate, 2 g/l of FeSO₄.7H₂O, sterile-filtered) wereadmixed with 5 ml of trace salt solution (0.15 g of Na₂MoO₄.2H₂O, 2.5 gof H₃BO₃, 0.7 g of CoCl₂.6H₂O, 0.25 g of CuSO₄.5 H₂O, 1.6 g ofMnCl₂.4H₂O, 0.3 g of ZnSO₄.7H₂O were made up to 1 l with water,sterile-filtered).

Solution 3 was made up as follows: 12.5 g of glucose were made up to 100ml with water and sterilized.

Solution 4 was made up as follows: 25 g of KH₂PO₄, 50 g of K₂HPO₄, 25 gof NaH₂PO₄ and 50 g of Na₂HPO₄ were made up to 500 ml with water andsterilized.

To the mixed medium (volume after addition of all solutions: 5 l) wereadded 100 ml of inoculation culture (in SVY medium (SVY medium: DifcoVeal Infusion broth 25 g, Difco Yeast extract 5 g, sodium glutamate 5 g,(NH₄)₂SO₄2.7 g were dissolved in 740 ml of H₂O and sterilized; 200 ml ofsterile 1M K₂HPO₄ (pH 7) and 60 ml of sterile 50% glucose solution wereadded (final volume 1 L))) of Bacillus subtilis PA668-2A and the culturewas fermented at 43° C. with vigorous stirring at a gas-introductionrate of 12 l/min. This strain is described according to the annex U.S.Ser. No. 60/262,995.

In the course of 48 h, approximately 4.3 l of a sterile aqueous glucosesolution were added. The solution was made up as follows: 5 kg ofglucose.H₂O and 3.3 g of CaCl₂.2H₂O were admixed with 2.1 kg ofdeionized water and sterilized for 30 min. Then, 11 ml of trace saltsolution (for composition see above) and 55 ml of citrate-iron solution(for composition see above) were added 73 ml of sterile-filtered 375 g/lsodium glutamate solution were then added.

The fermentation was carried out under glucose-limiting conditions.During the fermentation the pH was kept at 7.2 by adding 25% strengthammonia solution or 20% strength phosphoric acid. Ammonia actedsimultaneously as nitrogen source for the fermentation. The speed ofrotation of the agitator element was controlled to keep the dissolvedoxygen content to 20% of the saturation value. The foaming wascontrolled by occasional adding of the antifoam Tego KS 911. Afterhalting the addition of the carbon source, the fermentation wascontinued until the dissolved oxygen content (pO₂) had reached a valueof 95% of the saturation value. The fermentation was then ended. Thecells were separated off by centrifugation. Residual cells in thesupernatant were destroyed thermally by sterilization. The destructionwas demonstrated by plating out. After separation and sterilization, theD-pantothenate concentration was 29.6 g/l. Similarly, fermentationbroths can also be produced which have β-alanine-feed-free pantothenicacid-titers greater than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85 and greater than 90 g/l.

EXAMPLE 5

700 ml of the fermentation broth from Example 4 were pumped from bottomto top through a 1.4 l glass column in which was situated approximately1 l of the strongly basic ion exchanger Lewatit MP 500 (in the OH⁻form). The flow rate was 20 ml/min. The column was then washed at thesame pumping rate with approximately 2000 ml of deionized water. Thissolution contained cations in the form of NH4⁺, Ca²⁺, K⁺, Mg²⁺ and Na⁺ions. The pantothenic acid bound to the ion exchanger was then elutedusing approximately 2000 ml of a 5% strength calcium chloride solution.The eluate contained pantothenic acid in the form of calciumD-pantothenate, chloride, sulfate and phosphate ions of differingvalencies.

This aqueous calcium D-pantothenate solution was dried in a Niro Minorlaboratory spray dryer. The tower inlet temperature was about 200° C.,and the tower outlet temperature was 85-90° C. The atomization wasperformed using a two-fluid nozzle at a pressure of 4 bar. Powderingagent was not added. The pulverulent product had a specification of(data in % by weight):

-   Water content: 2%-   D-pantothenate: 24%-   Ammonium: 0.05%-   Potassium: 0.09%-   Sodium: 0.02%-   Magnesium: <0.01%

EXAMPLE 6

1000 ml of the fermentation discharge from Example 4 were pumped througha bed (volume about 1 liter) of the ion exchanger Lewatit MP 500 (in theCl⁻ form) and washed with about 2 l of water. The flow rate wascontrolled to approximately 20 ml/min.

At the same flow rate, 2 1 of a 10% strength calcium chloride solutionwere pumped through the ion exchanger. A solution was eluted which had aD-pantothenate content of 11 g/l. Cations in the form of ammonium,potassium, magnesium or sodium ions were not detected. The phosphate ionconcentration had been decreased to 7.7% compared with the startingsolution before the ion exchanger.

A 75 ml sample of the resultant calcium D-pantothenate solution was thendried by evaporating off the water on a rotary evaporator and 3.3 g of alight-brown calcium D-pantothenate powder were obtained which had acontent of 25% D-pantothenate. This powder does not have a tendency tostick together and has good product properties.

EXAMPLE 7

Of the filtrate obtained from Example 4, 1500 ml were passed through abed (1 liter) of the ion exchanger Lewatit MP500 (in the OH⁻ form). Theion exchanger was washed with water. The flow rate was approximately 20ml/min. At the same flow rate, approximately 2 liters of anapproximately 5% strength hydrochloric acid solution were passed throughthe ion exchanger.

The eluate was collected and adjusted to a pH of approximately 7 using20% strength milk of lime (suspension of calcium hydroxide in water).The eluate was then filtered once more through a paper filter and, asfiltrate, 2174 g of an aqueous calcium D-pantothenate solution having acontent of 19 g/l of D-pantothenate was obtained.

A 75 ml sample of the resultant calcium D-pantothenate solution was thendried by evaporating off water on a rotary evaporator and 5.7 g of abrown calcium D-pantothenate powder which had a content of 25%D-pantothenate were obtained. This powder does not have a tendency tostick together and has good product properties.

EXAMPLE 8

Of the filtrate obtained from Example 4, 1500 ml were passed through abed (1 liter) of the ion exchanger Lewatit MP500 (in the OH⁻ form). Theion exchanger was washed with water. The flow rate was approximately 20ml/min.

At the same flow rate, approximately two liters of an approximately 4%strength calcium hydroxide suspension were passed through the ionexchanger. The eluate was collected and adjusted to a pH ofapproximately 7. The aqueous calcium D-pantothenate solution has aD-pantothenate content of 21 g/l.

A 75 ml sample of the resultant calcium D-pantothenate solution was thendried by evaporating off water on a rotary evaporator and 5.6 g of abrown calcium D-pantothenate powder which has a D-pantothenate contentof 28% were obtained. This powder does not have a tendency to sticktogether and has good product properties.

1. A process for preparing D-pantothenic acid and/or salts thereof,which comprises a) fermenting at least one bacterium from theBacillaceae family which produces D-pantothenic acid and in which thebiosynthesis of pantothenic acid (pan) and/or isoleucine/valine (ilv) isderegulated and which forms at least 2 g/L of salts of D-pantothenicacid by fermentation in a culture medium, b) passing theD-pantothenate-containing fermentation solution through an anionexchanger and thereby binding the D-pantothenic acid to the anionexchanger, remaining compounds, including monovalent cations, passingthrough the anion exchanger, c) removing the D-pantothenic acid from theanion exchanger by eluting the D-pantothenate bound to the anionexchanger in the form of calcium and/or magnesium D-pantothenate by asolution containing inorganic or organic calcium salts and/or magnesiumsalts and/or inorganic calcium bases and/or magnesium bases and/ororganic calcium bases and/or magnesium bases, or eluting the boundD-pantothenate in the form of free D-pantothenic acid using an HCIsolution, d) optionally adding calcium base and/or magnesium base to setthe free D-pantothenic acid-containing eluate to a pH of 3-10, asolution being obtained which contains calcium and/or magnesiumpantothenate and e) subjecting the eluate or the solution containingcalcium pantothenate and/or magnesium pantothenate to drying and/orformulation wherein no free β-alanine and/or β-alanine salt is fed tothe culture medium.
 2. The process as claimed in claim 1, wherein thebacterium is of the genus Bacillus.
 3. The process as claimed in claim1, wherein, in step a) a content of D-pantothenic acid and/or saltsthereof of at least 10 g/l of culture medium is formed.
 4. The processas claimed in claim 1, wherein, in step b), the content of monovalentcations is reduced to a concentration of ≦1 g/kg of solution.
 5. Theprocess as claimed in claim 1, wherein the elution solution in step c)comprises acidic inorganic and/or organic calcium salts and/or magnesiumsalts, and/or basic inorganic and/or organic calcium salts and/ormagnesium salts.
 6. The process as claimed in claim 1, wherein, in stepc), the inorganic salts are calcium and/or magnesium halides.
 7. Theprocess as claimed in claim 1, wherein, in step c), the inorganic basesand/or salts are calcium hydroxide, calcium carbonate, calcium oxide,magnesium hydroxide or magnesium carbonate.
 8. The process as claimed inclaim 1, wherein, in step c), the organic salts are calcium and/ormagnesium formate, acetate, propionate, glycinate or lactate.
 9. Theprocess as claimed in claim 1, wherein, in step d), the pH of thesolution is set to 5-10.
 10. The process as claimed in claim 1, wherein,in step d), the pH of the solution is set to 5-9.
 11. The process asclaimed in claim 1, wherein, for neutralization, calcium hydroxide,calcium carbonate, calcium oxide, magnesium hydroxide and/or basicmagnesium carbonate is added in the form of a solid and/or as aqueoussuspension to the solution in step d).
 12. The process as claimed inclaim 1, wherein an aqueous suspension comprising 2-55% by weight ofcalcium hydroxide is added to the solution in step d).
 13. The processas claimed in claim 1, wherein an aqueous suspension comprising 2-65% byweight of calcium carbonate is added to the solution in step d).
 14. Theprocess as claimed in claim 1, wherein an aqueous suspension comprising20-60% by weight of magnesium hydroxide is added to the solution in stepd).
 15. The process as claimed in claim 1, wherein an aqueous suspensioncomprising 2-25% by weight of basic magnesium carbonate is added to thesolution in step d).
 16. The process as claimed in claim 1, wherein, instep d) or step e), a suspension is obtained which contains calciumpantothenate and/or magnesium pantothenate.